DNA binding shifts the redox potential of the transcription factor SoxR

Electrochemistry measurements on DNA-modified electrodes are used to probe the effects of binding to DNA on the redox potential of SoxR, a transcription factor that contains a [2Fe-2S] cluster and is activated through oxidation. A DNA-bound potential of +200 mV versus NHE (normal hydrogen electrode) is found for SoxR isolated from Escherichia coli and Pseudomonas aeruginosa. This potential value corresponds to a dramatic shift of +490 mV versus values found in the absence of DNA. Using Redmond red as a covalently bound redox reporter affixed above the SoxR binding site, we also see, associated with SoxR binding, an attenuation in the Redmond red signal compared with that for Redmond red attached below the SoxR binding site. This observation is consistent with a SoxR-binding-induced structural distortion in the DNA base stack that inhibits DNA-mediated charge transport to the Redmond red probe. The dramatic shift in potential for DNA-bound SoxR compared with the free form is thus reconciled based on a high-energy conformational change in the SoxR–DNA complex. The substantial positive shift in potential for DNA-bound SoxR furthermore indicates that, in the reducing intracellular environment, DNA-bound SoxR is primarily in the reduced form; the activation of DNA-bound SoxR would then be limited to strong oxidants, making SoxR an effective sensor for oxidative stress. These results more generally underscore the importance of using DNA electrochemistry to determine DNA-bound potentials for redox-sensitive transcription factors because such binding can dramatically affect this key protein property

Preliminary data on the structure and potential of the Tocomar geothermal field (Puna plateau, Argentina).

AbstractThis study presents new stratigraphic, structural and hydrogeological data on the Tocomar geothermal volcanic area (Puna plateau, Central Andes, NW Argentina), together with preliminary geochemical and magnetotelluric data.The main geothermal reservoir is located within the fractured Pre-Palaeozoic–Ordovician units. The reservoir is recharged by meteoric waters. Geothermal fluids upwell where main regional structures intersect secondary structures associated with the development of the Tocomar basin. Preliminary data indicate a reservoir temperature of ∼ 200° C and a local geothermal gradient of ∼ 130° C/km associated with the Quaternary volcanic activity in the Tocomar area

The Promoter of Rv0560c Is Induced by Salicylate and Structurally-Related Compounds in Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a major global health threat. During infection, bacteria are believed to encounter adverse conditions such as iron depletion. Mycobacteria synthesize iron-sequestering mycobactins, which are essential for survival in the host, via the intermediate salicylate. Salicylate is a ubiquitous compound which is known to induce a mild antibiotic resistance phenotype. In M. tuberculosis salicylate highly induces the expression of Rv0560c, a putative methyltransferase. We identified and characterized the promoter and regulatory elements of Rv0560c. PRv0560c activity was highly inducible by salicylate in a dose-dependent manner. The induction kinetics of PRv0560c were slow, taking several days to reach maximal activity, which was sustained over several weeks. Promoter activity could also be induced by compounds structurally related to salicylate, such as aspirin or para-aminosalicylic acid, but not by benzoate, indicating that induction is specific to a structural motif. The −10 and −35 promoter elements were identified and residues involved in regulation of promoter activity were identified in close proximity to an inverted repeat spanning the −35 promoter element. We conclude that Rv0560c expression is controlled by a yet unknown repressor via a highly-inducible promoter

Differential Gene Expression by RamA in Ciprofloxacin-Resistant Salmonella Typhimurium

Overexpression of ramA has been implicated in resistance to multiple drugs in several enterobacterial pathogens. In the present study, Salmonella Typhimurium strain LTL with constitutive expression of ramA was compared to its ramA-deletion mutant by employing both DNA microarrays and phenotype microarrays (PM). The mutant strain with the disruption of ramA showed differential expression of at least 33 genes involved in 11 functional groups. The study confirmed at the transcriptional level that the constitutive expression of ramA was directly associated with increased expression of multidrug efflux pump AcrAB-TolC and decreased expression of porin protein OmpF, thereby conferring multiple drug resistance phenotype. Compared to the parent strain constitutively expressing ramA, the ramA mutant had increased susceptibility to over 70 antimicrobials and toxic compounds. The PM analysis also uncovered that the ramA mutant was better in utilization of 10 carbon sources and 5 phosphorus sources. This study suggested that the constitutive expression of ramA locus regulate not only multidrug efflux pump and accessory genes but also genes involved in carbon metabolic pathways

Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation

Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins

Natural and anthropogenic features influencing water quality in NE Buenos Aires, Argentina

9 páginas, 5 figuras, 5 tablas, 2 mapas.-- Trabajo presentado en el VI ALHSUD- XXXII AIH Congreso Mar del Plata 2002.This work study water quality of the Pergamino River watershed in the Undulated Pampa in the northeast of the Argentinean Province of Buenos Aires. Temperature, pH, specific conductivity, Na, K, Mg, Ca, SO42Cl; HCO3, NO3, Si, Ag, Al, As, B, Ba, br, Cd, Co, Cr, Cu, Fe, Hg, Li, Mn, Mo, Ni, P, Pb, Se, Tl, U, V, Zn, and the (omega 18) O and (omega2)H isotope ratios were monitored in eighteen sampling stations. Groundwater and surface water are of sodium bicarbonate type, evolving to sodium sulfate-chloride type in the case of surface water.This work has been partially funded through the Proyect T657 from University of Buenos Aires.Peer reviewe

Rapid Changes in Gene Expression Dynamics in Response to Superoxide Reveal SoxRS-Dependent and Independent Transcriptional Networks

Background. SoxR and SoxS constitute an intracellular signal response system that rapidly detects changes in superoxide levels and modulates gene expression in E. coli. A time series microarray design was used to identify co-regulated SoxRSdependent and independent genes modulated by superoxide minutes after exposure to stress. Methodology/Principal Findings. soxS mRNA levels surged to near maximal levels within the first few minutes of exposure to paraquat, a superoxideproducing compound, followed by a rise in mRNA levels of known SoxS-regulated genes. Based on a new method for determining the biological significance of clustering results, a total of 138 genic regions, including several transcription factors and putative sRNAs were identified as being regulated through the SoxRS signaling pathway within 10 minutes of paraquat treatment. A statistically significant two-block SoxS motif was identified through analysis of the SoxS-regulated genes. The SoxRS-independent response included members of the OxyR, CysB, IscR, BirA and Fur regulons. Finally, the relative sensitivity to superoxide was measured in 94 strains carrying deletions in individual, superoxide-regulated genes. Conclusions/ Significance. By integrating our microarray time series results with other microarray data, E. coli databases and the primary literature, we propose a model of the primary transcriptional response containing 226 protein-coding and sRNA sequences. From the SoxS dependent network the first statistically significant SoxS-related motif was identified